The long-term goal of the proposed research is to reveal the physiological functions and in vivo mechanisms of G protein-coupled receptor (GPCR) endocytosis and postendocytic trafficking. GPCRs are the largest family of membrane receptors that receive sensory stimuli and mediate responses to neurotransmitters, neuropeptides, hormones, cytokines and growth factors. Activity-dependent endocytosis of GPCR reduces receptor numbers on the cell surface and is an important feedback regulation on the receptor signaling. In addition to being sorted into lysosome for degradation, endocytosed GPCRs are more frequently recycled back to the plasma membrane. The process of endocytosis and recycling is required for many receptors to dissociate from the binding ligand so that they can receive new stimuli. Failure of this process has been implicated in drug tolerances such as that to morphine. Although a large body of works has elicited various mechanisms of receptor endocytosis in cultured cells, the studies on GPCR endocytosis in intact organisms are still limited. More importantly, it is unclear how the endocytosed receptors are recycled back to the cell surface. In addition, the specific physiological functions of endocytosis and recycling have yet to identify for each GPCR. The major light receptor Rh1 rhodopsin in Drosophila eye is a model molecule for genetic characterization of GPCR signaling and regulation. Recently we identified a null mutant of a gene that encodes a CUB- and LDLa-domain protein (CULD), and found that a large amount of endocytosed Rh1 protein was retained in the cell body of the mutant photoreceptor. Our preliminary studies suggest that this is due to a failure of Rh1 recycling. We propose to take advantage of this culd mutant and several additional new mutant flies to study the mechanisms and the regulations of Rh1 endocytosis and recycling, and to characterize their impacts on the visual sensory function. Using a combination of molecular genetic, biochemical and electrophysiological approaches, we will 1. Confirm that the CULD protein is required for the recycling of Rh1 in photoreceptor 2. Test the hypothesis that CULD interacts with Arr1 for the localization of Rh1 in the rhabdomere 3. Test the hypothesis that loss of CULD impairs the development of light sensitivity in photoreceptors 4. Test the hypothesis that LAP is involved in the Rh1 endocytosis 5. Test the hypothesis that the deglycosylation of Rh1 restricts its endocytosis 6 Screen for additional molecules involved in the recycling of Rh1. PUBLIC HEALTH RELEVANCE: G protein-coupled receptor (GPCR) proteins on the cell membrane mediate >80% of transmembrane signaling activities, and are the major targets for pharmaceutical drug designs. In this proposal we plan to use Drosophila rhodopsin Rh1, a light-stimulated GPCR, as a model to genetically characterize the mechanisms underlying the receptor endocytosis and recycling. These processes regulate the intensity of GPCR signaling, and have been implicated in clinical disorders such as retinal degenerations and opioid tolerance.